Malignant brain tumors represent one of the most refractory cancers to therapy and remain incurable. Gliomas represent the most common type of brain tumors that occur in various grades, with the patient's prognosis inversely proportional to the grade. The long-term objective of my laboratory is to understand the cellular and molecular mechanisms that underlie tumor invasiveness in human gliomas. My laboratory has been active in the study of proteases and the biology of brain tumors, and data generated so far have indicated that changes in proteases are correlated with the changes in the grade of the tumors. The hypotheses are: 1) Regulation of MMP-9 in combination with cathepsin B or uPAR in xenograft cells via a plasmid construct expressing a siRNA message for either MMP-9 and uPAR (pMU) or MMP-9 and cathepsin B (pMC) will inhibit tumor growth and invasion;and 2) Regulation of these molecules using bicistronic RNAi constructs in xenograft cell lines will decrease/increase several signaling pathway molecules involved in cell survival, adhesion, migration and proliferation. The Specific Aims to address these hypotheses are as follows: Specific Aim 1. Evaluate the effect of vectors expressing siRNA targeting MMP-9, uPAR and cathepsin B, or a combination of these, on glioma cell growth and invasion both in vitro and in vivo. 1a. Determine the effect of vectors expressing bicistronic siRNA against MMP-9 and uPAR (pMU) or MMP-9 and cathepsin B (pMC) on MMP-9, uPAR and cathepsin B levels in glioma xenograft cell lines. 1b. Investigate and compare the effect of pMU or pMC on the invasive behavior of human glioma xenograft cells in in vitro models with that of control/mock, EV (empty vector) and SV (scrambled vector). 1c. Determine the effect of pMU or pMC on pre-established intracranial tumor growth or invasiveness of two glioma xenograft cells (XG3, and XG4) in nude mice. Specific Aim 2. Determine the effect of pMU/pMC on the molecular mechanisms of proliferation, migration and apoptosis in glioma xenograft cell lines. 2a. Determine the effect of pMU and pMC constructs on adhesion and migration of glioma xenograft cell lines. 2b. Evaluate the effect of pMU/pMC on the molecular mechanism of proliferation in glioma xenograft cells. 2c. Evaluate the effect of pMU/pMC on the molecular mechanism of apoptosis. We anticipate that these results will substantially augment our understanding of how MMP-9, cathepsin B, and uPAR molecules are regulated;thus, information gained should be of help in developing new therapeutic approaches to treat glioblastomas.